Simultaneous determination of retinol, alpha-tocopherol and retinyl palmitate in plasma of premature newborns by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of retinol, alpha-tocopherol and retinyl palmitate in plasma. Plasma containing an internal standard (tocol) was deproteinized with ethanol, then extracted with n-hexane. The organic layer was removed and evaporated under a nitrogen stream, and chromatographed on a reversed-phase RP-18 column using a water/acetonitrile-ethyl acetate/2-propanol gradient solvent system over 15 min at 305 nm. The recovery exceeded 93%. The detection limit was 0.1 microgram/ml for retinol, 1.3 micrograms/ml for alpha-tocopherol and 0.95 micrograms/ml for retinyl palmitate. The reproducibility, precision (expressed as coefficients of variation) and accuracy were less than 8% for all analytes. The small sample requirement, the simplicity of extraction, the short run-time and the good reproducibility make this procedure particularly useful for monitoring retinol and alpha-tocopherol supplementation in premature newborns.